WEEK 2 - NGS Technologies

Question 1

What are the fundamental requirements for any sequencing technology?

Answer 1

A sequencing method must:

Identify the position along a DNA molecule

Determine the base (A, T, G, or C) at that position

(For high-throughput methods) Link the sequence to the correct DNA fragment

Question 2

Describe the principle behind Sanger sequencing.

Answer 2

It uses a DNA polymerase, a primer, and a mix of normal dNTPs and chain-terminating fluorescently-labeled ddNTPs.
Incorporation of a ddNTP halts extension, producing fragments of varying lengths that are separated by capillary electrophoresis.
The color of the terminal base reveals the identity of each nucleotide.

Question 2

What distinguishes Sanger sequencing from next-generation sequencing (NGS)?

Answer 2

Sanger is a first-generation method that sequences one DNA fragment at a time with high accuracy, whereas NGS enables massively parallel sequencing of millions of fragments simultaneously, improving throughput but often sacrificing read length or initial accuracy.

Question 2

What is the Phred quality score used for?

Answer 2

It quantifies the accuracy of base calling. A score of Q30 corresponds to a 1 in 1000 chance of error (99.9% accuracy), indicating high-confidence data.

Question 2

Why does Sanger sequencing have a read length limit of ~1000 bp?

Answer 2

The ability to resolve single-nucleotide differences using electrophoresis decreases with fragment length, reducing accuracy for longer reads.

Question 3

What is pyrosequencing, and which technology uses it?

Answer 3

Used in 454 sequencing, pyrosequencing detects light emission resulting from pyrophosphate (PPi) release when a nucleotide is incorporated.
ATP sulfurylase converts PPi to ATP, which luciferase uses to generate light. The intensity of light correlates with the number of identical bases added.

Question 4

What is a major limitation of 454 pyrosequencing?

Answer 4

It struggles with homopolymeric regions (repeats of the same base), leading to imprecise length measurement and base calling.

Question 5

What makes Illumina sequencing the most widely used NGS platform?

Answer 5

High accuracy and reproducibility

Uses sequencing-by-synthesis with reversible terminators

Generates short reads (~150–300 bp), but allows deep coverage

Suited for applications like whole genome and transcriptome sequencing

Question 6

How does Illumina determine base identity?

Answer 6

Each nucleotide is added one at a time with a reversible terminator and a unique fluorescent label. After imaging, the terminator is removed to allow the next base to be incorporated.

Question 6

What differentiates PacBio and Oxford Nanopore from Illumina?

Answer 6

Both produce long reads (10 kb+), useful for resolving structural variants and repetitive regions

PacBio uses real-time synthesis detection with higher accuracy in its HiFi mode

Nanopore sequences DNA directly by measuring electrical changes as nucleotides pass through a protein nanopore

Question 6

What are the advantages of Oxford Nanopore sequencing?

Answer 6

Ultra-long reads (up to 1 million bases)

Real-time data acquisition

Portable devices (e.g. MinION) for field use

Ability to sequence RNA directly and detect base modifications like methylation

Question 7

What are real-world applications of Nanopore sequencing?

Answer 7

Epidemic tracking (e.g., Zika virus in Brazil)

Environmental biodiversity surveys

Rapid diagnostics for sepsis and kidney disease

On-site sequencing of marine organisms like reef fish and corals (used at JCU)